LOCUS       40924_47268        8543 bp DNA     circular SYN 13-MAY-2021
DEFINITION  measures Cas9 activity.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8543)
  TITLE     David Root CRISPR plasmids
REFERENCE   2  (bases 1 to 8543)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 8543)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8543
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    69..90
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        105..135
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    143..159
                     /label=lac operator
                     /note="The lac repressor binds to the lac operator to
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     167..183
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        204..222
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        250..476
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             477..657
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    704..829
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus
                     type 1"
     misc_feature    1322..1555
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1740..1784
                     /codon_start=1
                     /label=gp41 peptide
                     /note="antigenic peptide corresponding to amino acids 655
                     to 669 of the HIV envelope protein gp41 (Lutje Hulsik et 
                     al., 2013)"
                     /translation="KNEQELLELDKWASL"
     promoter        1962..2202
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     misc_feature    2364..2481
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination
                     sequence of HIV-1"
     promoter        2536..3040
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(3052..3071)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also
                     called puro-variant-R"
     primer_bind     3548..3568
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3688..3753
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             3760..4476
                     /codon_start=1
                     /label=EGFP
                     /note="enhanced GFP"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     misc_feature    4645..5233
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional
                     regulatory element"
     LTR             5305..5538
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5616..5750
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     rep_origin      5777..5912
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     promoter        complement(5933..5951)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5961..5977)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     rep_origin      6119..6574
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow
                     indicates direction of (+) strand synthesis"
     promoter        6600..6704
                     /label=AmpR promoter
     CDS             6705..7562
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      7736..8324
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     8478..8495
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
       61 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
      121 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
      181 gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg
      241 caagcttaat gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt
      301 agcaacatgc cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt
      361 ggtacgatcg tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca
      421 ctgaattgcc gcattgcaga gatattgtat ttaagtgcct agctcgatac ataaacgggt
      481 ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc
      541 ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg
      601 actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg
      661 gcgcccgaac agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac
      721 tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa
      781 aattttgact agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg
      841 ggggagaatt agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata
      901 taaattaaaa catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg
      961 cctgttagaa acatcagaag gctgtagaca aatactggga cagctacaac catcccttca
     1021 gacaggatca gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca
     1081 tcaaaggata gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa
     1141 caaaagtaag accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata
     1201 tgagggacaa ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag
     1261 gagtagcacc caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa
     1321 taggagcttt gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa
     1381 tgacgctgac ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt
     1441 tgctgagggc tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc
     1501 agctccaggc aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga
     1561 tttggggttg ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga
     1621 gtaataaatc tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa
     1681 ttaacaatta cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa
     1741 agaatgaaca agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca
     1801 taacaaattg gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt
     1861 taagaatagt ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat
     1921 tatcgtttca gacccacctc ccaaccccga ggggacccag agagggccta tttcccatga
     1981 ttccttcata tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga
     2041 ctgtaaacac aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt
     2101 agtttgcagt tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa
     2161 agtatttcga tttcttggct ttatatatct tgtggaaagg acgaaacacc ggtgaaccgc
     2221 atcgagctga gtttaagagc tatgctggaa acagcatagc aagtttaaat aaggctagtc
     2281 cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttgaattcta gatcttgaga
     2341 caaatggcag tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca
     2401 ggggaaagaa tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa
     2461 attacaaaaa ttcaaaattt tcgggtttat tacagggaca gcagagatcc actttggcgc
     2521 cggctcgagg gggcccgggt tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg
     2581 cggctgctct gggcgtggtt ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt
     2641 cttcacgtcc gttcgcagcg tcacccggat cttcgccgct acccttgtgg gccccccggc
     2701 gacgcttcct gctccgcccc taagtcggga aggttccttg cggttcgcgg cgtgccggac
     2761 gtgacaaacg gaagccgcac gtctcactag taccctcgca gacggacagc gccagggagc
     2821 aatggcagcg cgccgaccgc gatgggctgt ggccaatagc ggctgctcag cagggcgcgc
     2881 cgagagcagc ggccgggaag gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc
     2941 cctgttcctg cccgcgcggt gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt
     3001 cggctccctc gttgaccgaa tcaccgacct ctctccccag ggggatccac catgaccgag
     3061 tacaagccca ccgtgcgcct cgccacccgc gacgacgtgc ccagggccgt ccgcaccctc
     3121 gccgccgctt tcgccgacta ccccgccacc cgccacaccg tcgatcccga ccgccacatc
     3181 gagcgggtca ccgagctgca agaactcttc ctcacccgcg tcgggctcga catcggcaag
     3241 gtgtgggtcg ctgacgacgg cgccgcagtg gctgtctgga ccacacccga gagcgtcgaa
     3301 gctggggcgg tgttcgccga gatcggccct cgcatggccg agttgagcgg ttcccggctg
     3361 gccgctcagc aacagatgga aggcctcctg gctccacacc ggcccaagga gcccgcatgg
     3421 ttcctggcca ccgtcggcgt ctctcccgac caccagggca agggtctggg cagcgccgtc
     3481 gtgctccccg gagtggaggc tgccgagcgc gccggggtgc ccgccttcct ggagacctcc
     3541 gcaccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtggag
     3601 gtgcccgaag gaccacgcac ctggtgcatg acccgcaagc ccggtgccct gcagggatct
     3661 ggcgaagcca gacacaaaca gaaaattgtg aaacagactt tgaattttga ccttctcaag
     3721 ttggcgggag acgtggagtc caacccaggg cccgctagca tggtgagcaa gggcgaggag
     3781 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     3841 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     3901 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     3961 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     4021 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     4081 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     4141 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     4201 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     4261 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     4321 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     4381 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     4441 gccgggatca ctctcggcat ggacgagctg tacaagtccg gatcaggcgg aagcggcggg
     4501 agtgggtcac acggctttcc tcccgaagtt gaggagcagg atgatggcac cctgccgatg
     4561 tcctgcgctc aggaaagtgg catggacagg cacccagccg cgtgtgctag tgctagaatc
     4621 aacgtgtaaa cgcgttaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg
     4681 actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct
     4741 ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg
     4801 ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact
     4861 gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc
     4921 gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc
     4981 cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa
     5041 tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc
     5101 ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg
     5161 gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg
     5221 gccgcctccc cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
     5281 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc
     5341 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     5401 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     5461 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     5521 tgtggaaaat ctctagcagt acgtatagta gttcatgtca tcttattatt cagtatttat
     5581 aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg
     5641 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     5701 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc
     5761 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     5821 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     5881 ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt
     5941 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     6001 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     6061 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     6121 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     6181 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     6241 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     6301 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     6361 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     6421 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     6481 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     6541 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     6601 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     6661 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     6721 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     6781 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     6841 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     6901 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     6961 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     7021 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     7081 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     7141 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     7201 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     7261 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     7321 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     7381 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     7441 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     7501 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     7561 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     7621 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     7681 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     7741 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     7801 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     7861 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     7921 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     7981 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     8041 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     8101 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     8161 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     8221 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     8281 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     8341 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     8401 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     8461 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     8521 aaccgcctct ccccgcgcgt tgg
//
